Coronaviruses are a family of enveloped, single-stranded, positive-sense RNA viruses. The genomes of coronaviruses are the largest among all the RNA viruses, which has made their genetic manipulation a formidable problem. Our laboratory developed the first reverse genetic system for coronaviruses, called targeted RNA recombination. This system has been used to answer fundamental questions about viral protein structure and function, host species specificity, virion assembly, and the unusually complex mechanism of coronavirus genome RNA synthesis. Targeted RNA recombination is a powerful and versatile method that, in principle, should be applicable to all species of coronaviruses. The major object of this application is to develop and apply this system to the coronavirus that is the causative agent for Severe Acute Respiratory Syndrome (SARS-CoV). To accomplish this, we will construct an interspecies chimeric coronavirus that will have gained the ability to infect mouse cells. This virus, designated mSARS-CoV, will serve as the cornerstone for a host-range-based selection system for SARS-CoV reverse genetics. The tissue culture properties of mSARS-CoV and mutants of mSARS-CoV will be explored to gain insights into the potential roles of the eight unique genes in SARS-CoV that do not appear in other coronaviruses. The disease caused by these constructed viruses in the mouse host will be characterized and compared to that caused by the well-studied mouse coronavirus (MHV). Additionally, this genetic system will be used to answer basic questions about the components of SARS-CoV virion assembly and RNA synthesis and to create a mouse virus surrogate of SARS-CoV whose virion proteins are completely derived from MHV. The proposed work is expected to produce significant new information upon which more detailed future studies will be based. It will also generate valuable tools to test antiviral drugs, reveal targets for such drugs, and provide a means to manipulate SARS-CoV for vaccine design.